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A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S.

ABSTRACT: Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron-sulfur cluster that is necessary for activity. These findings provide an example of an iron-sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)-dependent amide bond formation in a natural product biosynthetic pathway.

SUBMITTER: Wang X

PROVIDER: S-EPMC8325364 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2.

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Characterization of the [3Fe-4S](0/1+) cluster from the D14C variant of Pyrococcus furiosus ferredoxin via combined NRVS and DFT analyses.

Project description:The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe-4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe-4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe-S clusters.

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A subclass of archaeal U8-tRNA sulfurases requires a [4Fe-4S] cluster for catalysis.

Project description:Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC + C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC + C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

| S-EPMC9825150 | biostudies-literature

Evidence that phosphorylation of iron regulatory protein 1 at Serine 138 destabilizes the [4Fe-4S] cluster in cytosolic aconitase by enhancing 4Fe-3Fe cycling.

Project description:Iron-sulfur cluster-dependent interconversion of iron regulatory protein 1 (IRP1) between its RNA binding and cytosolic aconitase (c-acon) forms controls vertebrate iron homeostasis. Cluster removal from c-acon is thought to include oxidative demetallation as a required step, but little else is understood about the process of conversion to IRP1. In comparison with c-acon(WT), Ser(138) phosphomimetic mutants of c-acon contain an unstable [4Fe-4S] cluster and were used as tools to further define the pathway(s) of iron-sulfur cluster disassembly. Under anaerobic conditions cluster insertion into purified IRP1(S138E) and cluster loss on treatment with NO regulated aconitase and RNA binding activity over a similar range as observed for IRP1(WT). However, activation of RNA binding of c-acon(S138E) was an order of magnitude more sensitive to NO than for c-acon(WT). Consistent with this, an altered set point between RNA-binding and aconitase forms was observed for IRP1(S138E) when expressed in HEK cells. Active c-acon(S138E) could only accumulate under hypoxic conditions, suggesting enhanced cluster disassembly in normoxia. Cluster disassembly mechanisms were further probed by determining the impact of iron chelation on acon activity. Unexpectedly EDTA rapidly inhibited c-acon(S138E) activity without affecting c-acon(WT). Additional chelator experiments suggested that cluster loss can be initiated in c-acon(S138E) through a spontaneous nonoxidative demetallation process. Taken together, our results support a model wherein Ser(138) phosphorylation sensitizes IRP1/c-acon to decreased iron availability by allowing the [4Fe-4S](2+) cluster to cycle with [3Fe-4S](0) in the absence of cluster perturbants, indicating that regulation can be initiated merely by changes in iron availability.

| S-EPMC2675999 | biostudies-literature

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